Results AP-2e is expressed in hypertrophic cartilage Previous studies demonstrated that the transcription factor AP-2e is expressed in chondrocytes and regu-lates gene expression of inte
Trang 1The family of activating enhancer-binding protein
(AP)-2 transcription factors regulate their target genes
through binding to the palindromic recognition
sequence 5¢-GCCN3GGC-3¢ or variations of this
GC-rich sequence within multiple gene promoters [1]
Both in vitro and in vivo data from AP-2 knockout
mice have shown their importance in numerous
physi-ological processes during development, cell cycle
regu-lation, and cell survival [1,2] The AP-2 family consists
of five members: AP-2a, AP-2b, AP-2c, AP-2d and
AP-2e [3–8] They all share a conserved basic-helix–
span–helix DNA-binding and dimerization domain
at their C-terminus, and a less conserved proline and
glutamine-rich transactivation domain at their N-ter-minus [9–11]
So far, the most recently identified AP-2 transcrip-tion factor, AP-2e, has been only poorly characterized [4,12] Expression of AP-2e was first described in the olfactory system [4], in skin, and in in vitro-cultured keratinocytes [12] Previously, we demonstrated that AP-2e is also expressed in chondrocytes, where it regu-lates the expression of integrin a10, the predominant collagen-binding integrin during cartilage development [13]
The axial skeleton is formed by a process named endochondral bone formation This complex process
Keywords
AP-2e; cartilage; differentiation;
osteoarthritis; transcriptional regulation
Correspondence
A.-K Bosserhoff, Institute of Pathology,
University of Regensburg,
Franz-Josef-Strauss-Allee 11, D-93053 Regensburg,
Germany
Fax: +49 941 944 6602
Tel: +49 941 944 6705
E-mail: anja.bosserhoff@klinik.
uni-regensburg.de
(Received 14 January 2009, revised 13
February 2009, accepted 18 February 2009)
doi:10.1111/j.1742-4658.2009.06973.x
Activating enhancer-binding protein (AP)-2e was previously described as a new regulator of integrin a10expression in cartilage In this study, we ana-lyzed the expression of AP-2e in differentiated chondrocytes and in human mesenchymal stem cells (HMSCs), which have been differentiated into chondrocytes in vitro AP-2e is predominantly expressed during the late stages of chondrocyte differentiation, mainly in early hypertrophic carti-lage, consistent with immunohistochemical stainings of mouse embryo sections Furthermore, osteoarthritic chondrocytes, resembling a hyper-trophic phenotype, have high AP-2e levels The AP-2e promoter harbors binding sites for the transcription factors AP-2a and Sox9 Both transcrip-tion factors strongly activate AP-2e expression in a cooperative manner in the chondrosarcoma cell line SW1353 The inhibition of Sox9 expression
by small interfering RNA resulted in decreased AP-2e expression In addition, direct interaction of Sox9 with the AP-2e promoter could be con-firmed by chromatin immunoprecipitation and electromobility shift assays This is the first study to prove the direct regulation of AP-2e by the transcription factor Sox9, and to indicate that AP-2e potentially has an important role as a modulator of hypertrophic cartilage
Abbreviations
AP, activating enhancer-binding protein; CD-RAP, cartilage-derived retinoic acid-sensitive protein; ChIP, chromatin immunoprecipitation; ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMSC, human mesenchymal stem cell; OA, osteoarthritis; SEM, standard error of the mean; siRNA, small interfering RNA.
Trang 2starts with the migration of undifferentiated
mesenchy-mal cells to regions that are destined to differentiate
into bones These progenitor cells condense and stick
together without increased proliferation [14,15] They
start to produce an extracellular matrix (ECM)
con-taining type I collagen, hyaluronic acid, tenascin, and
fibronectin [16–18] Subsequent differentiation of the
mesenchymal cells to chondrocytes causes a change in
ECM composition Chondrocytes express
cartilage-specific type II, type IX and type XI collagen,
proteoglycans, aggrecan, and cartilage-derived retinoic
acid-sensitive protein (CD-RAP), whereas expression
of type I collagen stops After further steps of
differen-tiation, chondrocytes become hypertrophic and express
increased levels of type X collagen and reduced levels
of type II collagen [19–21] Finally, osteoblasts
infil-trate into the cartilage and start to displace it with
mineralized bone
Sox9 represents an essential transcription factor of
the chondrogenic lineage, and regulates the expression
of chondrocyte-specific genes such as those encoding
type II collagen and CD-RAP [22,23] Sox9 belongs to
the HMG-box superfamily of transcription factors,
which bind in the minor groove of the DNA to the
consensus sequence (A⁄ T)(A ⁄ T)CAA(A ⁄ T)G [24]
Sox9 expression has been detected in all chondrogenic
progenitor cells and chondrocytes [25,26]
The cartilage, which mainly consists of chondrocytes
and ECM, serves as a protective layer for the joints
Degradation of the articular cartilage is a major
prob-lem in osteoarthritis (OA), a degenerative joint
disor-der, leading to destruction of the cartilage The onset
of this disease might be triggered by multiple factors
such as mechanical overload, defects in the
composi-tion of the ECM, or altered expression of transcripcomposi-tion
factors controlling the production of matrix molecules
[27]
Here, we analyzed AP-2e expression and its
regula-tion during cartilage differentiaregula-tion and in
osteo-arthritic chondrocytes Our data provide evidence that
AP-2e is directly regulated by the transcription factor
Sox9 and has a role in cartilage differentiation
Results
AP-2e is expressed in hypertrophic cartilage
Previous studies demonstrated that the transcription
factor AP-2e is expressed in chondrocytes and
regu-lates gene expression of integrin a10, which plays an
important role in cartilage development [13,28] To
determine the functional role of AP-2e in human
chon-drocytes, we used dedifferentiated chondrocytes and
human mesenchymal stem cells (HMSCs), and differ-entiated them either to chondrocytes or to osteoblasts [29] Figure 1A shows that AP-2e is highly expressed
in human chondrocytes and in chondrogenically differ-entiated HMSCs, as compared with untreated or osteoblastically differentiated HMSCs To further ana-lyze at which stages during chondrocyte differentiation the expression of AP-2e increases, we used an in vitro model system for HMSC differentiation into chondro-cytes established in our laboratory Marker genes for different stages of chondrogenesis, such as collagen type II, collagen type X, CD-RAP, and aggrecan, were analyzed to demonstrate differentiation stages [29] Using this model system, the expression of AP-2e mRNA was followed by quantitative real-time PCR over 40 days Interestingly, the expression of AP-2e increased relatively late during chondrogenic differenti-ation (Fig 1B) These stages correspond to the hypertrophic phase of chondrogenesis, which is charac-terized by increased expression of the hypertrophic marker gene type X collagen The expression of AP-2a, which is known to be expressed during carti-lage development [30], was analyzed as a control (Fig 1C) AP-2a expression increased early during chondrocyte differentiation and then remained at a moderate level The expression of the transcription factor Sox9, a key regulator of chondrogenesis, was also analyzed as a marker Sox9 was expressed early during chondrogenic differentiation, but its expression increased up to two-fold at later stages of differentia-tion, at around day 17 (Fig 1D)
To confirm AP-2e expression in hypertrophic regions of the developing cartilage, immunohistochem-ical stainings of tissue sections from 14.5-day-old and 17.5-day-old mouse embryos were performed using a specific polyclonal antiserum against AP-2e [31] (Fig 2A) AP-2e was detected in hypertrophic areas of the cartilage To verify the specificity, we stained sec-tions from AP-2e knockout mice (M Moser, unpub-lished data), and did not find any signal (Fig 2B) In parallel, we also analyzed Sox9 expression in these tissue sections Immunohistochemical staining with a specific Sox9 antibody demonstrated an increase of Sox9 in the early hypertrophic stages of cartilage development (Fig 2C)
AP-2e expression in osteoarthritic chondrocytes Osteoarthritic cartilage often resembles a hypertrophic phenotype [32,33] To address whether AP-2e expres-sion is altered in osteoarthritic chondrocytes in com-parison with differentiated chondrocytes, we quantified their mRNA expression, and detected significantly
Trang 3higher expression in osteoarthritic chondrocytes than
in differentiated chondrocytes (Fig 3A) Interestingly,
AP-2a expression was not increased in osteoarthritic
cartilage (Fig 3B) The expression of integrin a10, an
AP-2e target gene, was also measured, and was found
to be strongly upregulated in osteoarthritic
chondro-cytes as compared with differentiated chondrochondro-cytes
(Fig 3C) Furthermore, expression of Sox9 was
strongly increased in osteoarthritic chondrocytes as
compared with the control (Fig 3D)
Next, we wanted to confirm AP-2e expression in
cartilage from osteoarthritic patients To this end, we
performed immunohistochemical stainings of
osteo-arthritic cartilage with the AP-2e antiserum
Figure 3E shows AP-2e-positive cells within the
osteoarthritic cartilage tissue sections We also
ana-lyzed the expression of Sox9 in these tissue sections,
and found Sox9 expression in the osteoarthritic
carti-lage (Fig 3F)
AP-2a and Sox9 activate the AP-2e promoter
To obtain insights into the regulatory mechanisms
leading to the upregulation of AP-2e in the late stages
of cartilage differentiation and in osteoarthritic
chon-drocytes, we studied the AP-2e promoter One
thou-sand base pairs upstream of the translation start site
of the AP-2e gene were analyzed in detail to identify
binding sites for known transcription factors that might regulate AP-2e expression Two potential bind-ing sites for the transcription factor Sox9 at positions )973 ⁄ )970 and )448 ⁄ )445 and three putative AP-2a-binding sites at positions )322 ⁄ )312, )170 ⁄ )162 and )86 ⁄ )78 relative to the translation start site were identified (Fig 4A)
To test whether Sox9 or AP-2a regulates the expres-sion of AP-2e, the chondrosarcoma cell line SW1353 was transfected with expression constructs for each AP-2a and Sox9 or with both of them As a control, cells were transfected with expression constructs for Sox5 The expression of endogenous AP-2e mRNA was measured 24 h after transfection by quantitative real-time PCR Figure 4B shows that AP-2a or Sox9 transfection alone resulted in only low induction of AP-2e expression, but when AP-2a or Sox9 were trans-fected together, they strongly increased the expression
of AP-2e, up to 32-fold (Fig 4B) These data were confirmed by luciferase promoter assays First, a
302 bp construct of the AP-2e promoter sequence without a binding site for Sox9 (prom302) was cloned into a reporter gene plasmid containing a promoter-less luciferase gene SW1353 cells were transiently transfected with the AP-2e promoter construct, and luciferase activity was measured The 302 bp promoter construct showed no increased promoter activity as compared with the control (Fig 4C) In comparison
Fig 1 Expression of AP-2e in human chon-drocytes and human mesenchymal stem cells stimulated to undergo chondrogenic differentiation (A) Quantitative real-time PCR to measure the expression of AP-2e mRNA in human chondrocytes as compared with that in dedifferentiated chondrocytes, and in HMSCs stimulated to undergo chon-drogenic or osteoblastic differentiation in comparison with untreated cells (B–D) HMSCs were stimulated to undergo chondrogenic differentiation, and RNA was analyzed over 40 days The expression of AP-2e (B), AP-2a (C) and Sox9 (D) mRNA was analyzed using quantitative real-time PCR.
Trang 4with this, an AP-2e promoter construct of 604 bp
(prom604) containing binding sites for AP-2a and
Sox9 was clearly active in the cell line SW1353 as
com-pared with cells transfected with a control plasmid
Additional transfection of AP-2a or Sox9 expression
plasmids showed an increase of AP-2e promoter
activ-ity in SW1353 cells (Fig 4C) The cotransfection of
AP-2a and Sox9 further increased the promoter
activ-ity of AP-2e Transfection with an AP-2e expression
plasmid did not influence promoter activity, implying
that AP-2e does not regulate its own expression (data
not shown) A promoter construct with a mutation
within the Sox9-binding site (prom604mut) showed
decreased promoter activity as compared with the
wild-type 604 bp promoter construct Transfection
with an expression construct for Sox9 resulted in a
minor increase in promoter activity as compared with
the 604 bp wild-type construct The remaining activa-tion could be due to addiactiva-tional Sox9-binding sites within the AP-2e promoter that are less conserved
Sox9 is an activator of AP-2e expression
In the following studies, we focused on Sox9 as a regu-lator of AP-2e expression, because AP-2a is expressed
at a constant level during chondrocyte differen-tiation, and Sox9 is upregulated in the later stages
of differentiation Additionally, in OA, we found AP-2e and Sox9 to be upregulated but not AP-2a (Fig 3)
The influence of Sox9 on AP-2e expression was fur-ther analyzed using small interfering RNA (siRNA) against Sox9 SW1353 cells were transfected with con-trol siRNA, siRNAs against Sox9 (siSox9_2 and siSox9_5), or siRNAs against Sox5 (siSox5_1 and siSox5_4) as a second control First, Sox9 expression was measured after siRNA transfection (Fig 5A) A clear reduction of Sox9 expression could be shown after transfection with both Sox9 siRNAs, but not after transfection with control siRNA or siRNAs against Sox5 The reduction of Sox9 expression using siRNA strategies also caused a significant reduction of AP-2e expression (Fig 5B), suggesting that Sox9 is a positive regulator of AP-2e expression in chondrocytes
To demonstrate the direct interaction of Sox9 with the AP-2e promoter, chromatin immunoprecipitation (ChIP) assays were performed using SW1353 cells and
a specific Sox9 antibody DNA samples were analyzed
by PCR using specific primer pairs generating frag-ments spanning the first (Sox9_1) or the second (Sox9_2) Sox9-binding site of the AP-2e promoter Sox9 binding to both binding sites (Sox9_1 and Sox9_2) within the AP-2e promoter was observed
in vivo(Fig 5C)
Finally, the direct binding of Sox9 to the two Sox9-binding sites within the AP-2e promoter was con-firmed by electrophoretic mobility shift assays (EMSAs) Here, radioactively labeled oligonucleotides were used that harbored the Sox9-binding sites of the AP-2e promoter (Sox9_1 and Sox9_2) Incubation of
in vitro-synthesized Sox9 with the labeled oligonucleo-tides containing the Sox9-binding sites resulted in a strong DNA–protein interaction (Fig 5D, lanes 2 and 7) The specificity of these complexes was shown in competition studies using unlabeled oligonucleotides
in a 400-molar excess (Fig 5D, lanes 3 and 8) Incu-bation with a 400-molar excess of unlabeled oligonu-cleotides harboring a mutated Sox9-binding site did not lead to competition of the complexes (Fig 5D, lanes 4 and 9)
Fig 2 Expression of AP-2e and Sox9 in tissue slides of mouse
embryos (A) Immunohistochemical staining of AP-2e day 14.5 and
day 17.5 mouse embryos revealed strong signals in areas of
hyper-trophic cartilage (B) Tissue slides of an AP-2e knockout (ko) mouse
were stained as a control, and were clearly negative (C)
Immuno-histochemical staining of Sox9 in day 14.5 mouse embryos showed
Sox9 expression in the early stages of hypertrophic chondrocytes.
wt, wild-type.
Trang 5Recently, we showed that the transcription factor
AP-2e is a positive regulator of integrin a10expression
in chondrocytes [13] In this study, we wanted to
deter-mine the role of AP-2e expression during cartilage
development
Our results demonstrate that the transcription factor
AP-2e is expressed in human chondrocytes and in
HMSCs stimulated to undergo chondrogenic
differenti-ation To further investigate the time point of AP-2e
induction during chondrocyte differentiation,
chondro-genic differentiation of HMSCs was analyzed over a
time course of 40 days Expression data showed
increased expression of AP-2e in the late stages of
chondrocyte development At these stages of
differenti-ation, chondrocytes undergo a process of terminal
dif-ferentiation, by which they become hypertrophic and
express hypertrophic marker genes such as type X
col-lagen [34,35] Immunohistochemical staining of
embry-onic tissues at day 14.5 and day 17.5 confirmed clear
AP-2e expression in the hypertrophic cartilage Thus, AP-2e expression seems to correlate with hypertrophic cartilage differentiation
To determine how the increased expression of AP-2e
in hypertrophic chondrocytes is regulated, the sequence
of the AP-2e promoter was analyzed, and binding sites for the transcription factors AP-2a and Sox9 were identified Both transcription factors are known to play an important role in chondrocyte differentiation AP-2a is essential for skeletal development, and is expressed in limb buds during early embryogenesis, in the growth plate, and in chondrocytes of the joints [36] The AP-2a knockout mouse died at birth, with severe malformations of the craniofacial skeleton and defects in the development of the extremities [30,37]
We showed that moderate AP-2a levels might be important for AP-2e expression, as both Sox9 and AP-2a are needed to induce expression Thus, induc-tion of AP-2e expression is seen upon a further increase in Sox9 expression at later stages of chondro-cyte differentiation Therefore, we suggest that Sox9 is
Fig 3 Expression of AP-2e, AP-2a, integrin a10and Sox9 in differentiated chondrocytes as compared with that in osteoarthritic chondrocytes (A–D) Using quantitative real-time PCR analyses, the expression of AP-2e (A), AP-2a (B), inte-grin a 10 (C) and Sox9 (D) was measured in differentiated chondrocytes in comparison with osteoarthritic chondrocytes (n = 5) (E, F) Immunohistochemical staining of AP-2e (E) and Sox9 (F) in tissue slides of osteoarthritic cartilage revealed strong signals Black arrows indicate positively stained cells Data are given as mean ± SEM; *P < 0.05.
Trang 6an important regulator of AP-2e expression in
hyper-trophic chondrocytes and in osteoarthritis
The transcription factor Sox9 is known to be a
regu-lator of chondrogenesis It is expressed in all
chondro-genic progenitor cells and chondrocytes Sox9 is
essential for the early steps of chondrogenesis in
mes-enchymal condensation [38,39] In the later stages,Sox9
regulates the differentiation markers type II collagen
[22] and CD-RAP [23] Several groups have described
a reduction of Sox9 expression in hypertrophic
chon-drocytes [25,26], but in these studies no subdivision
was made into early and late hypertrophy Our
expres-sion analyses using quantitative real-time PCR and
immunohistochemical staining of Sox9 demonstrated
that Sox9 is expressed in early chondrogenic develop-ment and that expression is increased again at the beginning of the hypertrophic phase of differentiation, which is in accordance with other data [39,40] In detail, the study of Tchetina et al also proved that, in growth plates, Sox9 expression increased in the early hypertrophic zones of cartilage together with that of the hypertrophic marker gene type X collagen, and did not decrease until the late hypertrophic phase Thus, these experiments support our findings that Sox9 can positively regulate the expression of AP-2e in early hypertrophic chondrocytes
Using ChIP experiments and EMSAs, we confirmed the direct binding of the transcription factor Sox9 to
Fig 4 Promoter sequence of AP-2e, and regulation of AP-2e by AP-2a and Sox9 (A) Schematic illustration of the AP-2e promoter region Binding sites for the transcription factors AP-2a and Sox9 are indicated (B) SW1353 cells were transiently transfected with expression con-structs for AP-2a, Sox5 and Sox9, or with AP-2a and Sox9 The expression of AP-2e was measured using quantitative real-time PCR (C) Three hundred and two base pairs, 604 and 604 bp containing a mutated Sox9-binding site of the AP-2e promoter region were subcloned into pGL3-basic, and promoter activity was analyzed in SW1353 cells Additionally, expression constructs for AP-2a, Sox9 or both together were transiently transfected into SW1353 cells, together with the AP-2e promoter constructs, and promoter activity was measured pGL3-basic is set as 1 Data are given as mean ± SEM; *P < 0.05.
Trang 7the AP-2e promoter Further studies showed that Sox9
activates the promoter of AP-2e in cooperation with
AP-2a, resulting in an increase in AP-2e expression
Because AP-2a is expressed during chondrogenesis at a
constant level, we suppose that Sox9 is the crucial
factor in inducing AP-2e expression in the early
trophic phase of chondrocyte differentiation A
hyper-trophic phenotype is also characteristic for
osteoarthritic cartilage [32,33] Expression analyses of
osteoarthritic chondrocytes showed a strong increase
in AP-2e expression in these cells Sox9 expression is
also highly increased in osteoarthritic chondrocytes as
compared with differentiated chondrocytes, whereas
that of AP-2a is not
In summary, we demonstrated increased expression
of AP-2e in hypertrophic and osteoarthritic
chondro-cytes For the first time, we found that the
transcrip-tion factor Sox9 is a positive regulator of AP-2e
expression at the beginning of the hypertrophic
devel-opment of cartilage and of osteoarthritic chondrocytes
The dramatic increase in AP-2e expression and that of its target gene integrin a10 in OA suggests an impor-tant functional role of AP-2e in the development of hypertrophic chondrocytes To determine the role of AP-2e as a modulator of hypertrophy in cartilage, additional target genes of AP-2e, besides integrin a10, have to be determined
Experimental procedures
Cell culture
The chondrosarcoma cell line SW1353 was obtained from the American Type Culture Collection (ATCC, #HTB-94) Cells were maintained in high-glucose DMEM supple-mented with penicillin (400 UÆmL)1), streptomycin (50 lgÆmL)1), l-glutamine (300 lgÆmL)1), and 10% fetal bovine serum (Sigma, Deisenhofen, Germany), and split at
a 1 : 5 ratio every 3 days Primary chondrocytes were obtained from Cambrex (Iowa, IA, USA), and cultured as
Fig 5 Expression of AP-2e in SW1353 cells after silencing of Sox9 by siRNA transfection Expression levels of Sox9 (A) and AP-2e (B) were analyzed by quantitative real-time PCR after transfection of SW1353 cells with siRNAs (siSox9_2, siSox9_5), and compared with those in cells transfected with control siRNAs (control) or siRNAs against Sox5 (siSox5_1, siSox5_4) Data are given as mean ± SEM; *P < 0.05, ns, not significant Sox9 binds to the AP-2e promoter in vivo (C) A ChIP assay demonstrates the direct binding of Sox9 to the two Sox9-binding sites within the AP-2e promoter DNA samples of the ChIP reaction (Pol II, IgG, and Sox9) and the input DNA were used in PCR reactions with different primer pairs (GAPDH, negative control primers, Sox9_1 and Sox9_2) All PCR fragments could be detected in the input DNA sample A clear product of Sox9_1 and Sox9_2 was detected in the Sox9 ChIP DNA sample (D) EMSA to confirm the binding of Sox9 to the AP-2e promoter The contents of the reaction mixtures are marked above the image of the gel shift The Sox9 binding was shown using oligonucleotides spanning the two Sox9 regions Sox9_1 (lane 2) and Sox9_2 (lane 7) of the AP-2e promoter and in vitro-synthesized Sox9 protein For competition experiments, unlabeled wild-type oligonucleotides (lanes 3 and 8) and mutated oligonucleotides (lanes 4 and 9) were used Lanes 1 and 5 show the labeled oligonucleotides incubated without protein.
Trang 8suggested by the manufacturers The proliferating cells are
dedifferentiated in culture To differentiate these cells, they
were stimulated with transforming growth factor-b1
(10 ngÆmL)1) for 1 week
HMSCs from CellSystems (St Katharinen, Germany)
were cultivated in MSCGM medium (CellSystems) under a
humidified atmosphere of 5% CO2at 37C [41] To
stimu-late HMSCs to undergo either chondrogenic or osteoblastic
differentiation, cells were seeded in a 15 mL Falcon tube
and treated as previously described [29]
The cartilage samples used for immunohistology are of
human origin Cartilage was obtained from patients giving
informed consent following the standards of the Ethics
Com-mission of the University of Regensburg Full-thickness
car-tilage slices were aseptically dissected from healthy aspects of
femoral condyles of patients aged 50–76 years with
osteo-arthritis who had undergone total knee arthroplasty
OA chondrocytes were prepared from osteoarthritic
carti-lage slices obtained as described above, and are therefore
defined as osteoarthritic chondrocytes; no biochemical
mar-ker was used for characterization, except for the diagnosis
from the orthopedic surgeon according to accepted
ortho-pedic standards, which resulted in total joint replacement
surgery
RNA isolation, reverse transcription, and
quantitative real-time PCR
Total cellular RNA was isolated from cultured cells or
from tissues using the RNeasy kit (Qiagen, Hilden,
Germany), and cDNAs were generated by a reverse
trans-criptase reaction performed in a 20 lL reaction volume
containing 2 lg of total cellular RNA, 4 lL of 5·
first-strand buffer (Invitrogen, Groningen, the Netherlands),
2 lL of 0.1 m dithiothreitol, 1 lL of dN6-primer (10 mm),
1 lL of dNTPs (10 mm), and diethylpyrocarbonate⁄ water
The reaction mixture was incubated for 10 min at 70C,
200 U of Superscript II reverse transcriptase (Invitrogen)
were added, and RNAs were transcribed for 1 h at 37C
The reverse transcriptase was inactivated at 70C for
10 min, and the RNA was degraded by digestion with 1 lL
of RNaseA (10 mgÆmL)1) at 37C for 30 min
To precisely quantify the expression of cDNAs, the
real-time PCR LightCycler system (Roche, Mannheim,
Germany) was used as described previously [42,43] The
quantitative real-time PCR analysis of AP-2e, AP-2a, Sox9
and integrin a10 expression was performed using specific
primers: AP-2e-for, 5¢-GAAATAGGGACTTAGCTCTTG
G-3¢, and AP-2e-rev, 5¢-CCAAGCCAGATCCCCAACT
CTG-3¢ (annealing temperature 59 C); AP-2a-for, 5¢-GAT
CCTCGCAGGGACTACA-3¢, and AP-2a-rev, 5¢-GTTGG
ACTTGGACAGGGAC-3¢ (annealing temperature 60 C);
for, 5¢-CGAACGCACATCAAGACGA-3¢, and
Sox9-rev, 5¢-AGGTGAAGGTGGAGTAGAGGC-3¢ (annealing
temperature 58C); integrin alpha10-for, 5¢-CATGAGGTT
CACCGCATCACT-3¢, and integrin alpha10-rev, 5¢-AAGG CAAAGGTCACAGTCAAGG-3¢ (annealing temperature
64C) The expression ratios of the analyzed genes were calculated using an internal control standard curve of b-actin levels
Immunohistochemical staining
Paraffin sections of osteoarthritic cartilage and whole mouse day 14.5 and day 17.5 embryos were screened for AP-2e and Sox9 protein expression by immunohistochem-istry The tissues were fixed, and subsequently incubated with specific primary AP-2e antiserum [31] (1 : 200) or primary Sox9 antibody (Chemicon International Inc., Temecula, CA, USA) (1 : 100) overnight at 4C, with the secondary antibody (biotin-labeled anti-rabbit; DAKO, Hamburg, Germany) for 30 min at room temperature, and then with streptavidin-POD (DAKO) for 30 min Anti-body binding was visualized using AEC solution (DAKO) Finally, the tissues were counterstained with hemalaun solution (DAKO)
Plasmid constructs
Expression constructs for Sox9 and Sox5 were kind gifts from V Lefebvre (Department of Cell Biology, Cleveland Clinic, Cleveland, OH, USA) [44] An AP-2a expression plasmid was generated according to Moser et al [45] For analyses of the AP-2e promoter for putative tran-scription factor-binding sites, we screened approximately
1 kb of DNA of the upstream regulatory region, using the matinspector (Genomatix Software GmbH, Munich, Germany) We determined the start site of transcription
by extrapolation from cDNA clones and available expressed sequence tags, by analogy with the other four AP-2 iso-forms For generation of the AP-2e promoter constructs, the human genomic region was amplified by PCR with a 3¢-reverse primer (rev_promAP-2e, 5¢-GACAAGCTTGT AGGTGTGCACCAGCAT-3¢) in conjunction with two different 5¢-forward primers (for_promAP-2e_604, 5¢-GAC
CAGGC-3¢) To facilitate subcloning of the amplified frag-ment, the reverse primer contained a HindIII restriction site adaptor, and the forward primers contained an NheI site The PCR fragments and the luciferase expression vector pGL3-basic were digested separately with HindIII and NheI before ligation For generation of the promoter construct containing a mutated Sox9-binding site, site-directed muta-genesis with overlap extension was performed [46] For insertion of the mutated binding site, the following primers were used: mutSox9-447_for, 5¢-CCAGAAGGCGGCTCT GATTGCTGTGGGCTGAATTCACGC-3¢; and mutSox9-447_rev, 5¢-GCGTGAATTCAGCCCACAGCAATCAGAG CCGCCTTCTGG-3¢
Trang 9transfection solutions, according to the manufacturer’s
instructions The cells were harvested 24 h later, and RNA
was isolated
For measurement of luciferase promoter activity, each
cationic lipid⁄ plasmid DNA suspension was prepared by
mixing 0.2 or 0.5 lg of the luciferase reporter plasmid and
0.1 lg of the internal control plasmid pRL-TK with
trans-fection solutions, according to the manufacturer’s
instruc-tions The cells were harvested 24 h later, and the lysate
was analyzed for luciferase activity with a luminometer,
using Promega dual-luciferase assay reagent (Promega
Corporation, Madison, WI, USA) At least three
indepen-dent transfection experiments were performed for each
construct
siRNA transfection
The siRNAs against Sox9 (siSox9_2, siSox9_5) and the
control siRNAs (siSox5_1, siSox5_4 and control siRNA)
were synthesized by Qiagen Cells of the chondrosarcoma
cell line SW1353 were grown to 70–80% confluence in
culture dishes, and harvested in the proliferative growth
phase Cells were transfected with the HiPerFect
Trans-fection Reagent (Qiagen), according to the
manufac-turer’s protocol Cells were transfected in six-well
culture plates, and RNA was isolated 24 h after
trans-fection
ChIP assay
The ChIP assay was performed following the
manu-facturer’s instructions (ChIP-IT Express; Active Motif,
Carlsbad, CA, USA) SW1353 cells grown to 70–80%
confluence on three 15 cm plates were used for chromatin
isolation Samples were immunoprecipitated with a
specific Sox9 antibody (2 lg of anti-Sox9; Chemicon
International) An RNA polymerase II antibody was used
as a positive control, and an IgG antibody as a negative
control, following the protocol provided with the control
kit (ChIP-IT control Kit-human; Active Motif) DNA
samples from the ChIP experiments were used for
analy-sis by PCR PCR was performed on four DNA
tem-plates: the input DNA (1 : 5), DNA isolated through
RNA polymerase II ChIP (Pol II), DNA isolated through
the negative control IgG ChIP (IgG), and DNA isolated
through the Sox9 ChIP (Sox9) A control reaction with
no DNA template was also performed (H2O) Four sets
prom_rev, 5¢-CACTTCGCTCTCAGGCTTC-3¢ (213 bp fragment) PCR fragments were analyzed on a 1.5% agarose gel
Synthesis of Sox9 protein in vitro
Sox9 protein was synthesized by in vitro transcription– translation with the Sox9 expression vector and the TNT Quick Coupled Transcription⁄ Translation System (Promega Corporation, Madison, USA)
EMSA
The EMSA was based on the binding of Sox9 protein to a
32P-labeled oligonucleotide containing a Sox9-binding site Two double-stranded oligomeric binding sites for Sox9, specific for the AP-2e promoter (Sox9_1, 5¢-GCGG
5¢-CATGCCCACACTCAATCAGCCCAGGACCC-3¢) were generated The fragments correspond to the AP-2e pro-moter regions from )458 to )432 (Sox9_1) and from )985
to )957 (Sox9_2) upstream of the ATG The fragments were end-labeled with T4 polynucleotide kinase (Roche) and [32P]ATP[cP] (Amersham, GE Healthcare, Munich, Germany) Band shifts were performed by incubating
in vitro-synthesized Sox9 in the 5· mobility shift buffer [1 lg of poly(dI-dC)(dI-dC), 40% glycerol, 25 mm MgCl2,
1 mm EDTA, 25 mm dithiothreitol, 250 mm KCl, 25 mm Hepes⁄ KOH, pH 7.9) with the DNA probe for 10 min before separation on a 6% nondenaturing polyacrylamide gel For the competition studies, the cold oligonucleotides were added at a 400-fold molar excess and incubated for
10 min at room temperature before addition of the DNA probe DNAÆprotein complexes were resolved on a nondenaturing polyacrylamide gel at 250 V, 50 mA and
100 W for 1.5 h In vitro-synthesized protein was used to demonstrate the specificity of Sox9
Statistical analysis
Results are expressed as mean ± standard deviation (range) or percentage Comparison between groups was made using Student’s paired t-test A P-value < 0.05 was considered to be statistically significant All calculations were performed using graphpad prism software (GraphPad Software Inc., San Diego, CA, USA)
Trang 10This work was partly supported by a DFG grant
assigned to S Gra¨ssel (GR 1301 ⁄ 7-1)
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